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Thermotoga Maritima Chemical maturation of hydrogenases : an insight into artificial and biohybrid systems Giorgio Caserta To cite this version: Giorgio Caserta. Chemical maturation of hydrogenases : an insight into artificial and biohybrid sys- tems. Biomolecules [q-bio.BM]. Université Pierre et Marie Curie - Paris VI, 2016. English. NNT : 2016PA066701. tel-01631276 HAL Id: tel-01631276 https://tel.archives-ouvertes.fr/tel-01631276 Submitted on 9 Nov 2017 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Université Pierre et Marie Curie Ecole doctorale de Chimie Moléculaire de Paris-Centre Laboratoire de Chimie des Processus Biologiques CNRS UMR 8229 Collège de France, Paris Chemical maturation of hydrogenases: an insight into artificial and biohybrid systems Présentée par Giorgio CASERTA Pour l’obtention du grade de Docteur de l’Université Pierre et Marie Curie Spécialité Biochimie et Chimie Bio-inorganique Dirigée par le Professeur Marc Fontecave Présentée et soutenue publiquement le 7 Novembre 2016 Devant un jury composé de: Dr. Sandrine OLLAGNIER DE CHOUDENS, Directeur de Recherche CNRS Rapporteur Pr. Bruno GUIGLIARELLI, Professeur à Aix-Marseille Université Rapporteur Pr. Solange LAVIELLE, Professeur à UPMC Examinatrice Dr. Jean-Maurice MALLET, Directeur de Recherche CNRS Examinateur Dr. Mohamed ATTA, Directeur de Recherche CEA Grenoble Examinateur Pr. Marc FONTECAVE, Professeur au Collège de France Directeur de thèse This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Université Pierre et Marie Curie Ecole doctorale de Chimie Moléculaire de Paris-Centre Laboratoire de Chimie des Processus Biologiques CNRS UMR 8229 Collège de France, Paris Chemical maturation of hydrogenases: an insight into artificial and biohybrid systems Présentée par Giorgio CASERTA Pour l’obtention du grade de Docteur de l’Université Pierre et Marie Curie Spécialité Biochimie et Chimie Bio-inorganique Dirigée par le Professeur Marc Fontecave Présentée et soutenue publiquement le 7 Novembre 2016 Devant un jury composé de: Dr. Sandrine OLLAGNIER DE CHOUDENS, Directeur de Recherche CNRS Rapporteur Pr. Bruno GUIGLIARELLI, Professeur à Aix-Marseille Université Rapporteur Pr. Solange LAVIELLE, Professeur à UPMC Examinatrice Dr. Jean-Maurice MALLET, Directeur de Recherche CNRS Examinateur Dr. Mohamed ATTA, Directeur de Recherche CEA Grenoble Examinateur Pr. Marc FONTECAVE, Professeur au Collège de France Directeur de thèse This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Learn from yesterday, live for today, hope for tomorrow. The important thing is not to stop questioning. (A. Einstein) Summary Chemical maturation of hydrogenases: an insight into artificial and biohybrid systems There is a general agreement that the building of a sustainable H2 economy relies on the availability of cheap, abundant and efficient catalysts. Nature has provided attractive solutions, hydrogenases. However, these enzymes are difficult to produce and so far only few HydAs have been completely characterized showing diversity despite the same active site. This core, H-cluster, is composed of a [4Fe–4S] cluster bound via a Cys to a diiron complex which has 3 CO, 2 CN and an azadithiolate ligands. Recently, it has been showed that hydrogenases can be easily produced through the insertion of a biomimetic 2– [Fe2(adt)(CO)4(CN)2] complex inside the heterologously produced apo-enzyme, resulting in a full activation. Part of the PhD has been focused on the chemical maturation of new HydA from Megasphaera elsdenii and its truncated version, MeH-HydA, containing only the H- cluster. The assembly of all metal cofactors via the chemical reconstitution of the [Fe–S] 2– clusters and the maturation through the [Fe2(adt)(CO)4(CN)2] complex has been carried out. 2– Interestingly, HydF hybrids synthesized incorporating biomimetic [Fe2(xdt)(CO)4(CN)2] complexes onto the [4Fe–4S] cluster HydF protein, have a 6Fe core reminiscent of the H- cluster. HydFs from different organisms were purified and subsequently the [4Fe–4S] cluster has been reconstituted. For the first time, an X-ray structure of HydF with its [4Fe-4S] cluster has been obtained. The 6Fe cluster of HydF has been also prepared chemically with diiron complexes mimicking the active site of HydA. The metallo-cofactors have been spectroscopically characterized (EPR, FTIR, HYSCORE), hydrogenase activities evaluated. Keywords: [FeFe]-hydrogenase; artificial hydrogenase; chemical maturation; hydrogen evolution; iron-sulfur cluster; protein crystallography. Résumé Maturation chimique des hydrogénases: une étude des systèmes artificiels et biohybrides Le développement d’une économie basée sur l’hydrogène implique l’utilisation de catalyseurs efficaces et peu chers. Pour cela on peut s’inspirer de la nature qui a produit des métalloenzymes, les hydrogénases. On considère que la maturation est réalisée en deux étapes. Dans un premier temps, les clusters [4Fe–4S] sont assemblés par les systèmes ISC/SUF. Puis trois maturases, HydE/F/G réalisent la biosynthèse du 2Fe sous-cluster pour synthétiser le cluster-H. Seules quelques hydrogénases à [FeFe] ont été caractérisées montrant une grande diversité alors qu’elles possèdent le même centre catalytique, le cluster-H. Ainsi, une partie de cette thèse a porté sur l’utilisation de la «maturation chimique» pour activer de nouvelles enzymes apo-HydA. L’hydrogénase provenant de Megasphaera elsdenii, et sa version tronquée, MeH- HydA, contenant seulement le domaine du cluster-H ont été étudiées grâce à cette technique. La stratégie mise en œuvre a été la reconstitution des cofacteurs métalliques par l’assemblage des clusters [4Fe–4S] et la maturation des enzymes par le complexe biomimétique 2– [Fe2(adt)(CO)4(CN)2] . Les hybrides HydF synthétisés en incubant la protéine contenant le cluster [4Fe–4S] 2– avec les complexes biomimétiques [Fe2(xdt)(CO)4(CN)2] possèdent un centre à 6 fers similaire au cluster-H. Cette grande similarité amène au dernier point traité dans cette thèse: la possible activité catalytique d’HydF en tant que «hydrogénase artificielle». Les hybrides de HydF ont été caractérisés et leur activité d’hydrogénase a été évaluée. De plus, une structure RX de la protéine contenant son cluster [4Fe-4S] a été obtenue. Mots-clés : [FeFe]-hydrogénases; hydrogénase artificielle; maturation chimique; production d’hydrogène; cluster fer-soufre; cristallographie des protéines. Table of contents Abbreviations and Acronyms 7 Statement 11 Chapter I: Introduction 13 Section A 13 1.1 Metalloprotein: metal ions in living systems 13 1.2 Iron-sulfur cluster proteins 13 1.3 Biosynthesis of Fe–S clusters 16 1.4 Hydrogenase enzymes 16 Section B 19 1.5 The metal center of [FeFe]-hydrogenases 19 1.5.1 A protein machinery for [FeFe]-hydrogenase maturation 20 1.5.2 The Hyd proteins 22 1.5.3 The HydF protein 22 1.5.3.1 HydF, a scaffold protein 23 1.5.3.2 HydF, an iron-sulfur protein 25 1.5.3.3 HydF, a nucleotide-binding protein 26 1.5.3.4 Structure of the HydF protein 27 1.5.4 The HydE protein 29 1.5.4.1 HydE, a radical-SAM enzyme 29 1.5.4.2 HydE: a second, non-essential, cluster 30 1.5.4.3 HydE partners 31 1.5.4.4 Structure of the HydE protein 31 1.5.5 The HydG protein 33 1.5.5.1 HydG catalyzes tyrosine conversion to CO and CN 33 1.5.5.2 HydG: a radical mechanism 34 1.5.5.3 HydG: an unprecedented [5Fe-4S] cluster 35 1.5.5.4 Structure of the HydG protein 37 1.5.5.5 HydG enzyme mechanism 38 1.5.6 Mechanism of maturation of [FeFe]-hydrogenases 40 1.5.7 Redox states of the H-cluster of [FeFe]-hydrogenases 42 1.5.8 Maturase-free Chemical maturation: a unique technological tool 44 1 Section C 53 1.6 Artificial hydrogenases 53 1.6.1 Artificial hydrogenases based on synthetic diiron complexes 54 1.6.1.1 Micelles 56 1.6.1.2 Dendrimers 56 1.6.1.3 Polymers 57 1.6.1.4 Oligo-/poly-saccharides 58 1.6.1.5 Metal-Organic-Frameworks 59 1.6.2 Ni-based artificial hydrogenases 60 1.6.3 Artificial hydrogenases based on synthetic cobalt complexes 63 Section D 69 Aim of the project 69 Results and discussion 73 Chapter II: The [FeFe]-hydrogenase from Megasphaera elsdenii 73 2.1 Expression and aerobic purification of MeHydA 74 2.2 [Fe–S] cluster reconstitution of apo-MeHydA 75 2.3 Static light scattering of apo-MeHydA 77 2.4 EPR spectroscopic characterization of the iron-sulfur MeHydA 78 2.5 Chemical maturation 79 2– 2.5.1 Incorporation of synthetic [Fe2(adt)(CO)4(CN)2] complex 79 2– 2.5.2 Incorporation of synthetic [Fe2(pdt)(CO)4(CN)2] complex 80 2.6 FTIR and EPR spectroscopic characterization of holo-MeHydA 81 2.7 Conclusion and future perspectives: biotechnological application? 85 Chapter III: A truncated form of M. elsdenii [FeFe]-hydrogenase 91 3.1 Strategy 1: Cys to Ser mutations 93 3.1.1 Expression and purification of CystoSerMeHydA construct 94 3.2 Strategy 2: MeH-HydA 96 3.2.1 Cloning experiment 96 3.2.2 Expression and aerobic purification of apo-MeH-HydA 97 3.2.3 [4Fe–4S]H cluster reconstitution of MeH-HydA apoprotein 99 3.2.4 Spectroscopic characterization of FeS-MeH-HydA
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